Antisense oligonucleotides (ODN) against Smad7 and uses thereof in medical field

ABSTRACT

The invention relates to antisense oligonucleotidic sequences (ODN) against Smad7 suitably modified, and their uses in medical field as therapeutic biological agents, in particular in the treatment of chronic inflammatory bowel disease, such as Crohn&#39;s disease and ulcerative colitis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/201,061, filed Jul. 1, 2016, which is a continuation of U.S.application Ser. No. 14/815,339, filed Jul. 31, 2015, (issued as U.S.Pat. No. 9,382,541) which is a continuation of U.S. application Ser. No.14/613,181, filed Feb. 3, 2015 (issued as U.S. Pat. No. 9,096,854),which is a continuation of U.S. application Ser. No. 14/144,029, filedDec. 30, 2013 (issued as U.S. Pat. No. 9,006,418), which is acontinuation of U.S. application Ser. No. 13/836,634, filed Mar. 15,2013 (issued as U.S. Pat. No. 8,648,186), which is a continuation ofU.S. application Ser. No. 13/332,134, filed Dec. 20, 2011 (nowabandoned), which is a continuation of U.S. application Ser. No.12/854,558, filed Aug. 11, 2010 (issued as U.S. Pat. No. 8,106,182),which is a continuation of U.S. application Ser. No. 12/264,058, filedNov. 3, 2008 (issued as U.S. Pat. No. 7,807,818), which is a divisionalof U.S. application Ser. No. 11/501,756, filed Aug. 10, 2006 (nowabandoned), which is a continuation-in-part of U.S. application Ser. No.10/551,643, filed Jul. 24, 2006 (issued as U.S. Pat. No. 7,700,757),which is the national phase of International Patent Application No.PCT/IT2004/000117, filed Mar. 8, 2004, which claims priority to ItalianPatent Application No. RM2003 A 000149, filed Apr. 2, 2003, the entiredisclosure of each of which is incorporated by reference herein for allpurposes.

The present invention relates to antisense oligonucleotides (ODN)against Smad7 and uses thereof in medical field.

Particularly the invention refers to Smad7 antisense ODN sequencessuitably modified, which show a surprising biological activity ofspecific inhibition of Smad7 expression and are therefore usable inmedical field as therapeutic biological agents, in particular in thetreatment of chronic inflammatory bowel disease (IBD).

Crohn's disease (CD) and ulcerative colitis (UC) are the major forms ofchronic inflammatory bowel disease in human. Both diseases are complexclinical entities, whose pathogenesis is strictly dependent on theinteraction between different genetic, environmental and immune factors.

Despite CD and UC show marked differences both on the pathophysiologicaland clinical level, the therapeutic approach to suffering patientsshares many common elements. Variability of the clinical presentation,of the type and the extension of the lesions, and of the kind ofcomplications influences the therapeutic choice, even though thepharmacological treatment would represent the first predominantapproach.

Salicylazosulfapyridine and 5-aminosalicylic acid are drugs of provenefficacy in the management of the mild form of IBD and in the remissionmaintenance therapy.

In the phases with moderate to severe activity and in the cases in whichthe general state is involved, it is necessary to turn to the use ofcorticosteroids. From the medium and long-term analysis of the mainworldwide case histories, it appears that clinical remission isobtainable only in two thirds of patients receiving corticosteroids, andonly in 50% of these patients it does not occur any relapse after drugssuspension.

The continuous administering of corticosteroids, beside inducing drugsdependence phenomenon, is worsened due to a very high risk of sideeffects.

Also immunosuppressive treatment, which often accompanies or replacescorticosteroidal therapy, does not always ensure phlogosis containmentand control of symptoms, and further has the disadvantages of numerouscontraindications and severe side effects (Podolsky, 2002).

The new drug generation that became available in the 1990's, arebiological agents. The more in-depth knowledge of IBD natural historyand of the main pathophysiological mechanisms has contributed to steermedical intervention in a concrete way. Thus, a development ofbiotherapies aimed at controlling specific inflammatory “pathways”occurred through the use of recombinant human proteins, monoclonalchimeric humanized antibodies and fusion proteins. Contextually, agentswhich have showed a better efficacy in CD treatment are monoclonalchimeric antibodies directed to block TNF-α, a pro-inflammatory cytokineoverproduced during IBD (Seegers et al., 2002). This compound, which isat present in phase IV of clinical trial, is effective in theinflammation containment in about 60-70% of the treated patients.Nevertheless, some side effects have been pointed out with aconsiderable frequency of incidence and recognizable in reactivation oflatent microbial infections, hypersensitivity phenomena and formation ofautoantibodies. The latter phenomenon could be based on the fact thatanti-TNF-α neutralizes the cytokine TNF-α which has numerous biologicalfunctions.

In addition to its inflammatory effect, TNF-α takes part also to thosemechanisms involved in the induction and maintaining of immunologicaltolerance. Therefore a block of TNF-α activity could paradoxicallyencourage excessive immunological reactions (Sandborn et al., 2002).

All these remarks suggest the need of new studies on animal models ofIBD through which it is possible to identify new active principles to beused in a better and durable treatment of such pathologies (Fiocchi,2001).

Anti-TNF-α treatment, as far as the other biotherapies, such as theadministration of anti-inflammatory cytokines, for example IL-10,represents a therapeutic extracellular approach aimed at controllingbiological effects of molecules secreted by inflammatory cells.

The study of the signal-transduction pathways activated by cytokineinteraction with their receptors has outlined the chance to use newtherapeutic strategies capable to modulate specifically and selectivelythe intracellular expression of important inflammatory and noninflammatory molecules.

Under normal conditions, the intestinal mucosa is the seat of a“physiological” inflammatory infiltrate, which is tightly controlled byvarious counter-regulatory mechanisms.

In relation to the above, an important role is played by TGF-β1, amultifunctional cytokine capable of regulating the growth,differentiation and activity of many immune and non immune cells.

Both in vitro and in vivo studies have demonstrated that TGF-β1 acts asa potent immunoregulator able to control mucosal intestinalinflammation, and that the inhibition of its activity results in thedevelopment of colitis which shows immunomorphological similarity withCD or UC (Powrie F. et al., 1996; Neurath M. F et al., 1996; LudvikssonB. R. et al., 1997).

In fact, TGF-β1 genes deficient mice display severe multifocalinflammatory responses, also involving the intestine, associated with anexcessive inflammatory cytokines production by numerous cell types,including T cells (Shull M. M. et al., 1992; Christ M. et al. 1994).

Similarly, inhibition of TGF-β1 signalling in mouse by expressing adominant negative mutant form of the TGF-β1 receptor RII, results in anenhanced susceptibility to develop experimental colitis (Hahm K. B. etal., 2001).

Finally, it was shown that specific inhibition of TGF-β1 signaling in Tcells by the expression of a dominant negative TGF-β receptor type IIcauses an autoimmune disease characterised by severe inflammatoryinfiltrations in lung and colon and the presence of circulatingautoimmune antibodies (Gorelik L. et al., 2000). These data indicatethat the loss of activity of a single anti-inflammatory molecule couldbe sufficient to alter intestinal homeostasis and to allow immuneresponses leading to tissutal damage.

TGF-β1 anti-inflammatory activity starts with the interaction of themolecule with a complex of heterodimeric transmembrane serine/threoninekinases receptors consisting of two subunits, named TGF-β1 R1 and TGF-β1R2 respectively. Upon TGF-β1 binding, the receptors rotate relativelywithin the above mentioned complex, resulting in a trans-phosphorylationprocess and subsequent activation of TGF-β1 R1 by the constitutivelyactive TGF-β1 R2 and capable of autophosphorylation.

The propagation of the TGF-β1-triggered signal to the nucleus ismediated by proteins belonging to the Smad family. Activated TGF-β1 R1directly phosphorylates Smad2 and Smad3 proteins, which become able tointeract with Smad4, thus enabling the complex Smad2-3/Smad4 totranslocate to the nucleus, where it participates to the transcriptionalcontrol of some genes (Heldin C-H. et al., 1997).

The role of Smad3 in the TGF-β1 anti-inflammatory activity was supportedby studies in animal models, which show that the deletion of theencoding gene for Smad3 is associated with diminished cellresponsiveness to TGF-β1, and with a related development of inflammatorydisease characterized by a massive infiltration of T-cells and pyogenicabscesses formation at gastrointestinal level (Yang X. et al., 1999).

Also other intracellular proteins, for example Smad7, belong to theSmads protein family. Such protein occupying TGF-β1 R1 interferes withthe binding of Smad2/Smad3 to the receptor, thus preventing thephosphorylation and the activation. Hence, an increased expression ofSmad7 protein is associated with an inhibition of the TGF-β1-mediatedsignaling (Hayashi H. et al., 1997).

The evaluation of the TGF-β1 expression in intestinal mucosa from IBDpatients shows that said molecule production is paradoxically enhancedin comparison to what can be proved in the gut of normal patients(Lawrance I C. et al., 2001).

In a recent article the author of the present invention shows thatmucosal samples from IBD patients are characterized by high levels ofSmad7 and by reduced levels of active Smad3, thus indicating that duringIBD the mechanism of TGF-β1-mediated signaling is compromised. Theauthor of the present invention further showed that selective Smad7abrogation by a specific antisense oligonucleotide5′-GTCGCCCCTTCTCCCCGCAGC-3′ (SEQ ID No 1) restores lamina propriamononuclear cells (LPMC) responsiveness to TGF-β1, resulting in adown-regulation of pro-inflammatory cytokine production, such as forexample, TNF-α.

Moreover, also ex vivo experiments carried out on intestinal mucosasamples from IBD patients showed that administration of Smad7 antisenseODN restores TGF-β1 signaling mechanism and allows a diminished cytokineproduction (Monteleone et al., 2001).

During IBD, intestinal mucosa is infiltrated with an high number of Tcells. These cells are regarded to be the main mediators of tissutaldamage acting in such diseases.

The increased number of T cells in the intestinal mucosa from IBDpatients is partly dependent on the resistance of such cells againststimuli inducing their death (apoptosis).

It is believed that the block of T cells apoptosis plays a key role inmaintaining the mucosal inflammatory response in IBD (Boirivant et al.,1999). Indeed, enhancing T cell death associates with a resolution ofthe intestinal inflammation. The exact mechanism underlying theresistance of T cells against apoptosis during IBD is not yet known,even if locally released cytokines seem to be involved.

Data from cell-culture in vitro experiments and in vivo studies indicatethat TGF-β1 can either prevent or trigger T cell death and that thecapacity of the factor to mediate both responses is site-specific (Han SH. et al., 1998; Arsura M. et al., 1996)

Smad3 knockout mice exhibit a massive increase in the number ofinflammatory cells at the intestinal level, thus suggesting a role forTGF-β1 in controlling intestinal T cell apoptosis at intestinal level(Yang et al., 1999).

Therefore Smad7 inhibition by the use of Smad7 synthetic antisense ODNmay represent a novel and acceptable “endogenous” biotherapeuticapproach to chronic inflammatory diseases, in particular to IBD, since,as above mentioned, it restores T cells responsiveness to TGF-β1.

Antisense oligonucleotides (ODN) are short synthetic oligonucleotidicsequences complementary to the messenger RNA (m-RNA) which encodes forthe target protein of the specific and aimed inhibition. Such sequences,hybridizing to the m-RNA, make a double-strand hybrid trait, and thisleads to the activation of ubiquitary catalytic enzymes, such as RNasesH, which degrade DNA/RNA hybrid strands that develop in nature totrigger DNA duplication, thus preventing protein translation.

The selection of the most suitable m-RNA regions and sequences tohybridize to the ODN has empirical characteristics even if ODNcomplementary to the transcriptional initiation region 5′ and to thesplicing regions usually result more effective. The design of aremarkable number of antisense ODN, after identifying possible targetsites, does not raise difficulties, thanks to the recent and advancedautomated synthesis technologies owned by specialized companies in suchfield.

On the contrary the identification of the more active ODN, for possibletherapeutic applications, requires a long-term screening work throughefficacy assays in quantitative test. In relation to the above,antisense ODN sequences against specific target, among which Smad7, arealready known (U.S. Pat. No. 6,159,697; assignee ISIS PharmaceuticalsInc.).

The use of antisense ODN both for in vitro and in vivo gene regulationis thwarted by some problems, such as, the difficulty to pass throughcellular membranes, due to the polianionic and then hydrophilic natureof these molecules, and the rapid enzymatic degradation.

To overcome these obstacles it is necessary to resort to chemicalmodification of the antisense ODN, such as, for example,phosphorothioation, as in the case of the above mentioned Smad7 specificsequence (Monteleone et al., 2001), or phosphoroamidation, that aresubstitutions of sulphur or nitrogen atoms in place of those oxygenatoms which are not the bridge atoms of the phosphodiester linkage.

As well as many biotechnological products, the demonstration of abiological activity points out a potential therapeutic activity.

Indeed ODN can be used either in the studies of both gene and proteinfunctions involved in the pathogenesis of different diseases or fortherapeutic purpose. Whereas in the former application field theantisense methodology was successful for the easiness of the guideprinciples, the shift from in vitro to in vivo experimentation is morecomplex, especially as regards pharmacokinetic, pharmacodynamic andtoxicological aspects of these new drugs (Maggi A., 1998).

For example the Smad7 antisense ODN used in the previous experimentscarried out by the author of the present invention (SEQ ID No 1), whichshows in vitro biological activity, could show an increased risk ofundesirable effects in vivo. In fact, such ODN contains two nucleotidicCG pairs which become CpG after phosporothioation, an essential processto enhance ODN stability. The latter are sequences endowed with apowerful stimulating activity of the immune system, therefore the use ofthe above mentioned ODN as such could made worse the course of anyimmunologic disease, Crohn's disease and ulcerative colitis included.

A similar therapeutic approach could not be hypothesized, especially inthe case of Crohn's disease, a pathology mediated by a particular classof T lymphocytes, named Th1, under the interleukin 12 stimulus. IndeedCpG molecules, as powerful inductors of the IL-12 synthesis, couldinduce a further development of Th1 cells.

In addition, in vivo administration of the antisense ODN containing CpGdinucleotides is accompanied by an increased risk of side effects, incomparison to oligonucleotides without CpG. In particular, it has beenproved an increased risk of hyperplasia of the reticuloendothelialsystem of the spleen, kidney and liver, as well as an increasedproliferation of hematopoietic cells (Agrawal S. et al., 2002).

Another problem in the use of ODN is bound to the side effects resultingfrom the action of the metabolites derived from the degradation of themolecule, which results quite susceptible to nuclease attack, since itis not protected at the 5′ and 3′ ends.

Therein the necessity of chemical modification of the phosphorothioateantisense ODN backbone to CpG pairs and to 5′ and 3′ ends. Neverthelessthe above said modifications of the ODN sequence could lead up to thereduction or the loss of the biological activity of inhibition of Smad7synthesis and, sometimes, even to the inversion of the desired activityboth in vitro and in vivo.

Likewise it may be important to dispose of experimental IBD modelssuitable for in vivo studies, which allow to enlarge the knowledge onthe mechanisms involving the loss of the regulation of the immunitaryresponse and their role in the onset of IBD pathology and on thepossibility to modulate or prevent such response, thus limitinginflammation progression at mucosal level. In relation to the above theTNBS-mediated colitis represents a spread and valid model of mucosalinflammation which shows striking immunomorphological similarities withhuman CD (Neurath M. et al., 2000).

In the light of the above, it would be desirable to dispose of newtherapeutic biological agents, like Smad7 antisense ODN, which areactive both in vitro and in vivo, for the treatment of IBD through an“endogenous” biotherapeutic approach.

The author of the present invention has now found suitably modifiedantisense ODN sequences which exhibit an higher in vivo biologicalactivity of inhibition of Smad7 expression in experimental models of IBDin comparison to their in vitro inhibitory activity, and also higherthan that of other known sequences showing the same modifications andtested on the same models.

In particular, the ODN sequences which exhibit an higher in vivobiological activity were designed according to the phosphorothioateantisense ODN sequence SEQ ID No 1 targeting the site 403 of the humanSmad7 RNA, used by the author of the present invention in the course ofprevious experiments.

In view of the potential and future use of such Smad7 phosphorothioateantisense ODN for the treatment of human pathologies said sequence wasmodified at CpG dinucleotides therein contained, hereinafter indicatedas XY, because of their already mentioned immunogenicity.

The study carried out by the author has allowed to test in vivo and invitro efficacy of different known and novel Smad7 antisense ODN andtheir possible toxicity, and to investigate if blocking Smad7 expressionresults in a resolution of mucosal inflammation in experimental modelsof IBD.

The above mentioned suitably modified antisense ODN sequences accordingto the present invention, in addition to an higher biological activityin vivo, showed a surprisingly absence of side effects in animals, inspite of what happens after the administration of other sequences duringthe course of the same study. Further, ODN sequences according to theinvention showed their efficacy to limit lymphocytic infiltration andthe ulterior inflammation propagation, that is an evidence not found forthe other antisense ODN sequences herein tested.

The role of Smad7 as biological target clearly appears from thesestudies in experimental models, together with the possible therapeuticeffects of its inhibition.

Furthermore, within the context of the present invention, another roleof Smad7 on the induction of T cell apoptosis during IBD has been found.In fact, through the use of some Smad7 antisense ODN, it has been showedthat TGF-β1 regulates intestinal T cell apoptosis and that a defectivefactor activity accounts for cell-resistance to apoptotic stimuli, whichare responsible for maintaining the mucosal inflammatory response.

Therefore the objects of the present invention are Smad7phosphorothioate antisense oligonucleotides up to 21 nucleotides inlength which comprise a portion of at least 10 nucleotides of thefollowing sequence (SEQ ID No 2):

5′-GTXYCCCCTTCTCCCXYCAG-3′

wherein X is a nucleotide comprising a nitrogenous base selected fromthe group consisting of cytosine, 5-methylcytosine and2′-O-methylcytosine and wherein Y is a nucleotide comprising anitrogenous base selected from the group consisting of guanine,5-methylguanine e 2′-O-methylguanine, provided that at least one of thenucleotides X or Y comprises a methylated nitrogenous base;

or the complementary sequence thereto.

Other objects of the present invention are the oligonucleotidicsequences of the different antisense oligonucleotide stereoisomers, suchas diastereoisomers and enantiomers, as to the phosphor atoms of theinternucleosidic linkage included in the sequence. Indeed theinternucleosidic linkage can be phosphorothioate or methylphosponate andin both cases the phosphor bound to four different chemical groupsrepresents a chiral centre.

Antisense oligonucleotides according to the present invention can haveat least one methylphosphonate nucleotide into the sequence, which isplaced, for example, either at only one of the 5′ or 3′ ends or at both5′ and 3′ ends or along the oligonucleotidic sequence.

In a preferred embodiment the methylphosphonate nucleotide can be eitherX or Y, in such a way that internucleosidic linkage is the linkagebetween said nucleotides.

Further modifications can be carried out to the 5′ and 3′ ends and/oralong the sequence of the antisense ODN to increase the stability of themolecule thus preventing the degradation by nucleases and reducing therisk of undesirable effects derived from the metabolite actions.

Antisense oligonucleotides according to the present invention can havefurther at least one nucleotide of the sequence which is a2′-O-methylribonucleotide 5′-monophosphate, which is placed, forexample, either at only one of the 5′ or 3′ ends or at both 5′ and 3′ends or along the oligonucleotidic sequence.

Further objects of the present invention are the above, said antisenseoligonucleotide wherein 2′-deoxyribonucleotides are replaced byribonucleotides and 2′-deoxythymidine is replaced by uridine in such away that antisense deoxyribonucleotidic sequences turn to thecorrespondent antisense ribonucleotidic sequences.

A preferred embodiment of the present invention is represented byantisense oligonucleotides having the sequence (SEQ ID No 3):

5′-GTXGCCCCTTCTCCCXGCAG-3′ wherein X is 5-methyl 2′-deoxycytidine5′-monophosphate. In a particular preferred embodiment, the inventionrelates to the above sequence further having a cytosine nucleotide at 3′end (SEQ ID No 16; 5′-GTXGCCCCTTCTCCCXGCAGC-3′).

Another preferred embodiment is represented by antisenseoligonucleotides having the sequence (SEQ ID No 4):

5′-ZTXGCCCCTTCTCCCXGCAZ-3′

wherein X is 5-methyl 2′-deoxycitidine 5′-monophosphate and Z is2′-deoxyguanosine methylphosphonate.

According to another aspect, a preferred embodiment of the presentinvention is antisense oligonucleotide having the sequence (SEQ ID No15):

5′-ZTXGCCCCTTCTCCCXGCAZC-3′

wherein X is 5-methyl 2′-deoxycytidine 5′-monophosphate and Z is2′-deoxyguanosine methylphosphonate.

Antisense ODN sequences according to the present invention can beadvantageously used in medical field; therefore further objects of thepresent invention are pharmaceutical compositions which comprise atleast one of the above disclosed antisense oligonucleotides as activeprinciple together with one or more pharmaceutically acceptableadjuvants and/or excipients, which are known to skilled person in thisfield.

Further the invention relates to the use of the aforesaid antisenseoligonucleotide sequences for the preparation of a medicament for thetreatment of the pathologies associated with Smad7 expression. Inparticular, such pathologies associated with Smad7 expression are IBD,such as, for example, CD and UC.

The present invention is now described, for illustrative but notlimitative purposes, according to its preferred embodiments, withparticular reference to the figures of the enclosed drawings, wherein:

FIG. 1 shows the effect of Smad7 ODN antisense(5′-MePGTMe-dCGCCCCTTCTCCCMe-dCGCAMePG-3′ (SEQ ID No 4) and sense on thepresence of CD3+ T lymphocytes in ex vivo organ cultures of mucosalexplants of CD patients after 40 hour treatment;

FIG. 2 shows the analysis of the expression of p-Smad2/Smad3 complex andof the total Smad2/Smad3 complex in LPMC isolated from the intestine ofTNBS-treated mice (TNBS), untreated (Unt), treated with ethanol (EtOH)as controls;

FIG. 3 shows the analysis of the Smad7 expression in LPMC isolated fromthe intestine of TNBS-treated mice (TNBS), untreated (Unt), treated withethanol (EtOH) as controls;

FIG. 4 shows the percentage changes in weight of mice with TNBS-inducedcolitis treated or not with Smad7 antisense oligonucleotideMePGTMe-dCGCCCCTTCTCCCMe-dCGCAMePGC (SEQ. ID No 15) or with a control(sense); the figure is representative of three separate experimentswherein fourteen mice for each group have been studied;

FIG. 5 shows macroscopic aspect of the colon extracted from a mouse withTNBS-induced colitis and from a mouse with TNBS-induced colitis treatedwith Smad7 antisense oligonucleotide (SEQ. ID No 15); the figure isrepresentative of three separate experiments wherein fourteen mice foreach group have been studied;

FIG. 6 shows histological aspect of a colon section from mice withoutcolitis or with TNBS-induced colitis treated or not with Smad7 antisenseoligonucleotide (SEQ. ID No 15) or with a control (sense); the figure isrepresentative of three separate experiments wherein fourteen mice foreach group have been studied. Magnification 40×;

FIG. 7, panel A, shows the inhibition of murine Smad7 protein by aspecific antisense oligonucleotide. Transfection of RAW cells with afluorescent (F)-labelled Smad7 antisense oligonucleotide was confirmedby microscopy (upper panel) and flow cytometry (lower panel) (numbersindicate the % of transfected cells). Right inset shows the inhibitionof Smad7 protein by Smad7 antisense (AS) but not sense (S)oligonucleotide after 48 hours of culture. Cells were starved overnightand then transfected with the oligonucleotides in the presence oflipofectamine (L). Smad7 was assessed by Western blotting using totalproteins. Panel B shows orally administered fluorescent-labelled Smad7antisense oligonucleotide in mice with TNBS-colitis is taken up by bothepithelial and lamina propria mononuclear cells in the proximal smallintestine, terminal ileum and colon after 8 hours of administration. Nostaining is seen in mice with TNBS-colitis at time 0. Panel C showsrepresentative Western blots showing down-regulation of Smad7 andenhanced p-Smad3 in the colon of mice with TNBS-colitis and treated withSmad7 antisense (AS) oligonucleotide. Mice with TNBS-colitis were orallyadministered with 125 μg/mouse of either AS or sense oligonucleotide atday 1, killed at day 4, and total extracts used for Smad analysis. Oneof 6 representative experiments analysing in total 12 mice per group isshown;

FIG. 8, panel A, shows the effects of different doses of orallyadministered Smad7 antisense (AS) oligonucleotide on weight of mice withTNBS colitis. Mice were left untreated (naïve) or treated with TNBS, and1 day after the induction of colitis administered or not with Smad7sense (125 μg/mouse) or AS (50, 125 or 250 μg/mouse). Data indicate thecumulative mean weight data from 2 separate experiments. In eachexperiment, each group consisted of at least four mice. Bars representS.E.M. Panel B shows weight changes of mice with TNBS-colitis after asingle administration of Smad7 AS or sense oligonucleotide. Either theAS or sense oligonucleotide (125 μg/mouse) was orally administered 1 dayafter (day 1) the induction of colitis, and weight of each mouse wasthen daily recorded. Each point represents the cumulative mean weightdata from 4 separate experiments. In each experiment, each groupconsisted of at least four mice. Bars represent S.E.M. Panel C showshistologic evaluation of TNBS-colitis in mice treated or not with Smad7antisense (AS) or sense oligonucleotide. Photomicrograph (×20) of anH&E-stained paraffin section of a representative colon from micebelonging to each group. Severe mucosal mononuclear cell infiltrate anddisruption of the normal crypt architecture with epithelial ulcerationand loss of goblet cells is evident in the colon of mice with TNBSeither left untreated or treated with the sense oligonucleotide. Incontrast, only small scattered areas of cellular infiltration is seen inthe colon of mice treated with the Smad7 AS oligonucleotide. Thephotomicrographs are representative of 3 separate experiments in whichat least 4 mice per group were studied;

FIG. 9, shows the inhibition of Smad7 protein by a Smad7 antisense (AS)oligonucleotide in mice with TNBS-colitis results in decreased tissueexpression of IFN-γ(A), IL-12 (both p40 and p70, panels B and C). Oneday after the TNBS-colitis induction, mice were either left untreated ortreated with 125 μg/mouse of Smad7 AS or sense oligonucleotide. Micewere then sacrificed at day 4, and colonic samples used for extractingtotal proteins. Cytokines were analyzed by ELISA and data are expressedas pg/μg total proteins. Each point represents the value of cytokine incolonic samples taken from a single mouse. Horizontal bars indicate themedian value. Induction of TNBS-colitis results in a significantincrease in the production of IFN-γ in comparison to naive and ethanol(EtOH)-treated mice. Consistently, both IL-12/p40 and IL-12/p70 areincreased during TNBS-colitis in comparison to control mice. Oraladministration of Smad7 antisense but not sense oligonucleotide to micewith TNBS-colitis reduces IFN-γ as well as IL-12/P40 and IL-12/p70synthesis;

FIG. 10, panel A, shows representative Western blots showing bothdown-regulation of Smad7 and enhanced p-Smad3 in mice with oxazolone(oxa)-colitis and treated with Smad7 antisense (AS) or senseoligonucleotide. Mice were orally administered with 125 μg/mouse ofeither AS or sense oligonucleotide the day after the induction ofcolitis (day 1), then killed at day 3, and total proteins extracted fromthe colon and used for Smad analysis. One of 3 representativeexperiments analysing in total 7 mice per group is shown. B.Quantitative data of either Smad7/β-actin or p-Smad3/Smad3 as measuredby densitometry scanning of Western blots. Values are expressed inarbitrary units (a.u.) and indicate mean±S.E.M. of all experiments.Panel C shows the histologic analysis of the colons from mice withoxazolone-induced colitis, either left untreated or treated with Smad7antisense (AS) or sense oligonucleotide. Photomicrographs of H&E-stainedparaffin section of distal colon (×20) from mice at day 3 after theinduction of colitis are shown. A severe transmural inflammation withbowel wall thickening, epithelial cell ulceration and loss of gobletcells are seen in the colon of mice with oxazolone-colitis either leftuntreated or treated with the sense oligonucleotide. In contrast, aminimal mononuclear cell infiltration of the lamina propria withmaintenance of the epithelial cell architecture is present in the colonof mice treated with the Smad7 AS oligonucleotide. Right inset shows thehistological score of colonic sections from mice with oxa-inducedcolitis, either left untreated or treated with Smad7 antisense (AS) orsense oligonucleotide. Data indicate the mean±SD of 2 separateexperiments analysing in total 7 mice per each group. Treatment of micewith Smad7 AS but not sense significantly reduces the colonicinflammation;

FIG. 11, panel A, shows photomicrograph (×20) of an H&E-stained paraffinsection of a representative colon of 2 mice (1 naïve and 1 withTNBS-induced colitis). Severe mucosal mononuclear cell infiltration anddisruption of the normal crypt architecture with epithelial ulcerationand loss of goblet cells is evident in the mouse with relapsingTNBS-colitis. In this experiment, mice were sacrificed one day after thelast TNBS injection. Percent of animals harboring mild, moderate, andsevere colitis are shown as bar graphs. Panel B shows enhanced Smad7 anddecreased p-Smad3 expression in mice with relapsing colitis. The dayafter the last (fourth week) TNBS injection mice were killed, andcolonic total proteins analysed for Smad molecules by Western blotting.Right inset. High Smad7 is seen in purified lamina propria T lymphocytes(CD3+) and CD3-negative LPMC from mice with relapsing TNBS (T)-colitisin comparison to ethanol (E)-treated mice. Cells were isolated from thecolon of 3 mice per each group. Panel C shows oral administration ofSmad7 antisense (AS) oligonucleotide leads to an early recover in weightin mice with TNBS-induced chronic colitis. Colitis was induced asindicated in material and methods and the day after the last TNBSinjection mice were allocated to receive either Smad7 AS or senseoligonucleotide. Weight changes were then daily recorded. Each pointrepresents the cumulative mean weight data from two experiments in which8 mice per group were considered. Bars represent S.E.M. Right inset.Administration of Smad7 antisense to mice with relapsing TNBS-colitisreduces Smad7 expression both in colonic CD3-positive and CD3-negativeLPMC. Cells were isolated from the colon of 3 mice per each group. PanelD shows histologic evaluation of chronic TNBS-colitis in mice treatedwith Smad7 antisense (AS) or sense oligonucleotide. Photomicrograph(×20) of an H&E-stained paraffin section of a representative colon frommice belonging to each group. A moderate mucosal mononuclear cellinfiltration, dilatation of the colon, and hyperplastic lymphoidfollicles are seen in sections from mice treated with senseoligonucleotide, whereas only a minimal inflammatory infiltrate is notedin the colon of mice treated with the Smad7 AS. The photomicrographs arerepresentative of experiments in which 5 mice per group were studied.Percent of animals with no evidence of colitis or harboring mild,moderate, and severe inflammation are shown as bar graphs.

EXAMPLE 1: STUDY ON THE EFFECT OF SMAD7 ANTISENSE OLIGONUCLEOTIDESACCORDING TO THE PRESENT INVENTION ON INTESTINAL T CELLS APOPTOSIS

Materials and Methods

Synthesis of Antisense ODN

All the Smad7 antisense ODN were synthesized by MWG Biotech AG (MWGBiotech S.r.l., Florence) employing standard automated techniques withan automated DNA synthesizer using standard phosphoroamidite chemistryprotocols (Lesiak K. et al., 1993; Xiao W. et al., 1996).

Oligonucleotides containing 5-methyl-2′-deoxycitidine (5-Me-dC) weresynthesized according to known synthesis methods (Sanghvi et al., 1993)using commercially available phosphoroamidites, whereas synthesis ofmodified oligonucleotides containing methylphosphonate groups (MeP) wasaccomplished using known protocols (Maier M A. et al., 2002).

The purification of the oligonucleotidic molecules has been carried outHPSF technology, developed by MWG Biotech. Such purification method hasrevealed an high efficiency since it allows removing failure sequencessynthesized during the automated chemical synthesis process, such as,for example, n-1, n-2, n-x sequences, that standard purification classicmethods are not capable to remove.

The above mentioned technology, besides enabling to obtain 100% of thedesired length sequences without undesirable failure products, allowsavoiding next desalting operation, since the purified sequences are freeof both salt and metal ions.

Given the absence of any salt, oligonucleotides were eventuallyanalysized by MALDI-TOF mass spectrometry techniques according tostandard protocols (Guerlavais T. et al., 2002; Ragas J A. et al.,2000). Then oligonucleotides were sterilized and the resulting solutionwas quantified as optical density (OD) by UV/visible spectrophotometer.Finally the molecules were resuspended in sterile PBS1× before using.

All the used antisense ODN target Smad7 m-RNA sites which have 100%homology between human and mouse. In all the following oligonucleotidesthe internucleoside linkage is a phosphorothioate linkage.

The antisense ODN sequences being used in the present study have beendesigned according to the phosphorothioate antisense ODN sequence5′-GTCGCCCCTTCTCCCCGCAGC-3′ (SEQ ID No 1) targeting the site 403 of thehuman Smad7 m-RNA, used by the author of the present invention in thecourse of previous experiments (Monteleone et al., 2001).

The Smad7 antisense ODN sequence5′-MePGTMe-dCGCCCCTTCTCCCMe-dCGCAMePG-3′ (SEQ ID No 4) targets the site403 of the human Smad7 m-RNA. This is a mixed-backbone oligonucleotidewherein the cytosine belonging to CpG pairs of SEQ ID No 1 were replacedby 5-methylcytosine (herein indicated as Me-dC). In addition,methylphosphonate linkages were placed at the ends of the molecule(herein indicated as MeP).

The Smad7 antisense ODN sequence 5′-GTTTGGTCCTGAACATGC-3′ (SEQ ID No 5)targets the site 294 of the human Smad7 m-RNA.

Mucosal samples were taken from resection specimens of 6 patients withmoderate-to-severe CD and 4 patients with severe UC. In addition,intestinal mucosal samples were taken from 10 unaffected IBD patientsundergoing colectomy for colon carcinoma (ethical approval was obtainedby local committee). LPMC were prepared using the DTT-EDTA-collagenaseprocedure and resuspended in RPMI 1640 (Sigma-Aldrich S.r.l., Milan)supplemented with a serum replacement reagent HL-1 (Biowhittaker,Wokingham, UK).

Cells were cultured in the presence and absence of TGF-β1(Sigma-Aldrich, final concentration ranging from 1 to 5 ng/ml) and after48 hours of incubation were analyzed for the level of apoptosis.

In other experiments, LPMC isolated from IBD patients intestine wereresuspended in RPMI 1640 supplemented with HL-1 and cultured in thepresence and absence of the above mentioned Smad7 antisense ODN (SEQ IDNo 4, SEQ ID No 5), and in the presence of a control senseoligonucleotide (both used at a concentration of 2 μg/ml). After 24hours, an aliquot of LPMC was used for extracting proteins and evaluateSmad7 expression. The remaining cells were extensively washed andresuspended in RPMI 1640 plus HL-1 and cultured in the presence orabsence of TGF-β1 (5 ng/ml) for 48 hours and then analyzed forapoptosis.

Analysis of Apoptosis by Flow Cytometry

Apoptosis was analyzed by propidium iodide (PI) staining followed byflow cytometry.

Cells were washed, incubated for 15 minutes at 37° C. in 5 μlribonuclease A (0.6 μg/ml, 30-60 Kunitz units, Sigma-Aldrich), and thenchilled on ice. Propidium iodide (100 μg/ml) was added before analysisby flow cytometry.

T cells were identified using a specific monoclonal anti-CD3 antibody(DAKO Ltd., Cambridgeshire, UK).

Protein Extraction and Western Blot Analysis

LPMC were homogenized and total proteins were extracted in buffer Acontaining 10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mM EDTA and 0.2 mM EGTA.Buffer was supplemented with 1 mM dithiothreitol (DTT), 10 μg/mlaprotinin, 10 μg/ml leupeptin and 1 mM phenylmethanesulphonyl fluoride(all reagents from Sigma-Aldrich).

Smad7 protein was analyzed using a specific rabbit anti-human Smad7antibody (1:400 final dilution, Santa Cruz Biotechnology, Inc., CA;USA). Goat anti-rabbit antibodies conjugated to horseradish peroxidase(Dako Ltd) were used at 1:20.000 final dilution to detect primaryantibody binding and immunoreactivity was visualized with achemiluminescence kit (Pierce, Rockford, Ill., USA).

Organ Culture

Mucosal explants taken from the surgical specimens of patients werecultured in the presence or absence of Smad7 antisense ODN (SEQ ID No 4,SEQ ID No 5; both used at a final concentration of 10 μg/ml) for 40hours.

As negative control, a mucosal explant was cultured in the presence ofSmad7 sense ODN.

At the end of the culture, mucosal explants were collected and used foranalyzing the number of lamina propria T lymphocytes byimmunohistochemistry.

For this purpose, mucosal sections were prepared and stained with amonoclonal anti-CD3 antibody (DAKO). Goat anti-mouse antibodiesconjugated to alkaline phosphatase (DAKO) were used to detect primaryantibody binding.

Results

The results obtained in the different experiments show how TGF-β1enhanced, dose-dependently, apoptosis of T lymphocytes isolated from theintestine of normal subjects.

Table 1 shows the percentage of apoptotic T lymphocytes after 48 hoursof culture. Numbers are the results of 4 separate experiments in which Tcells isolated from the intestine of four normal subjects were used.

TABLE 1 Exp. 1 Exp. 2 Exp. 3 Exp. 4 Unstimulated 18% 17% 19% 23% TGF-β1(0.2 ng/ml) 22% 24% 23% 25% TGF-β1 (1 ng/ml) 31% 33% 28% 31% TGF-β1 (5ng/ml) 33% 34% 32% 37%

In contrast, T lymphocytes isolated from four IBD patients showed apartial resistance to the TGF-β1-induced apoptosis signal as shown inthe results reproduced in Table 2 which shows the percentage ofapoptotic T lymphocytes after 48 hours of culture.

TABLE 2 Exp. 1 Exp. 2 Exp. 3 Exp. 4 Unstimulated 11% 10%  9% 7% TGF-β1(0.2 ng/ml) 12%  9%  8% 5% TGF-β1 (1 ng/ml) 10% 11% 11% 8% TGF-β1 (5ng/ml) 16% 13% 14% 15% 

In particular, from the analysis of data shown in Table 2 no meaningfulincrease in apoptosis was seen when T cells from IBD patients werecultured in the presence of either 0.2 ng/ml or 1 ng/ml TGF-β1concentration. In contrast, stimulation of T cells from IBD patientswith 5 ng/ml TGF-β1 resulted in a small increase in apoptosis.

Treatment of T lymphocytes isolated from IBD patients with the Smad7antisense ODN SEQ ID No 4 restored the cell responsiveness to TGF-β1,resulting in enhanced cell apoptosis, as shown in percentage values of Tlymphocytes reproduced in Table 3. Data refer to four separateexperiments in which T cells isolated from the intestine of four IBDpatients, were cultured with medium alone (unstimulated) or pre-treatedwith medium and sense (control) or antisense oligonucleotides overnightand then stimulated with TGF-β1 (1 ng/ml).

TABLE 3 Exp. 1 Exp. 2 Exp. 3 Exp. 4 Unstimulated 10%  9%  8% 7% Medium11%  9%  8% 5% ODN Sense 12% 10% 10% 8% ODN Antisense 33% 32% 23% 19% 

Furthermore, using ex vivo organ culture, the author of the presentinvention demonstrated that treatment of IBD biopsies with Smad7antisense ODN according to the present invention significantly decreasedthe number of mucosal CD3+ T cells, as shown in the immunohistochemistryof FIG. 1. The latter shows that the treatment with the antisense ODNreduces the number of mucosal CD3+ T cells.

Together these observations suggest the possibility that high Smad7level plays a crucial role in prolonging T cell survival, therebycontributing to the propagation of local inflammation in IBD.

Thus, blocking Smad7 could represent a promising strategy to controlmucosal inflammation in these condition.

EXAMPLE 2: IN VIVO AND IN VITRO STUDIES ON THE EFFECTS OF THEADMINISTRATION OF SMAD7 ANTISENSE AND SENSE OLIGONUCLEOTIDES INEXPERIMENTAL MODELS OF TNBS-INDUCED COLITIS

Material and Method

All the Smad7 antisense and sense ODN were synthesized by MWG BiotechS.r.l. (Firenze) employing the standard techniques previously described.

The used antisense ODN target Smad7 m-RNA sites which have 100% homologybetween human and mouse. In all the following oligonucleotides theinternucleoside linkage is a phosphorothioate linkage. All the followingsequences were used in the experiments carried out on the experimentalinduced-colitis models.

The Smad7 antisense ODN SEQ ID No 1 (5′-GTCGCCCCTTCTCCCCGCAGC-3′)targets the site 403 of the human Smad7 m-RNA already used by the authorof the present invention in the course of experiments published in aprevious article (Monteleone et al., 2001).

For the further study concerning the role of Smad7 on the regulation ofT cell apoptosis in LPMC isolated from the intestine of IBD patients thefollowing antisense oligonucleotide sequences SEQ ID No 4 e SEQ ID No 5were used.

The Smad7 antisense ODN sequence5′-MePGTMe-dCGCCCCTTCTCCCMe-dCGCAMePG-3′ (SEQ ID No 4) targets the site403 of the human Smad7 m-RNA. This is a mixed-backbone oligonucleotidewherein the cytosine belonging to CpG pairs in the position 3 and 16 ofSEQ ID No 1 were replaced by 5-methylcytosine (indicated as Me-dC). Inaddition, methylphosphonate linkages were placed at the ends of themolecule (indicated as MeP).

The Smad7 antisense ODN sequence 5′-GTTTGGTCCTGAACATGC-3′ (SEQ ID No 5)targets the site 294 of the human Smad7 m-RNA. The internucleosidelinkages included therein are phosporothioate linkages.

The Smad7 antisense ODN sequence 5′-GTTTGGTCCTGAACAT-3′ (SEQ ID No 6)targets the site 296 of the human Smad7 m-RNA.

The Smad7 antisense ODN sequence 5′-GTTTGGTCCTGAACATG-3′ (SEQ ID No 7)targets the site 295 of the human Smad7 m-RNA.

The Smad7 antisense ODN sequence 5′-AGCACCGAGTGCGTGAGC-3′ (SEQ ID No 8)targets the site 577 of the human Smad7 m-RNA.

The Smad7 antisense ODN sequence 5′-MePAGCACMedCGAGTGMedCGTGAGCMeP-3′(SEQ ID No 9) targets the site 577 of the human Smad7 m-RNA. This is amixed-backbone oligonucleotide wherein the cytosine in the position 6and 12 of SEQ ID No 8 were replaced by 5-methylcytosine. In addition,methylphosphonate linkages were placed at the ends of the molecule.

The Smad7 antisense ODN sequence 5′-CGAACATGACCTCCGCAC (SEQ ID No 10)targets the site 233 of the human Smad7 m-RNA.

The Smad7 antisense ODN sequence 5′-Me-d CGA ACA TGA CCT CMe-d CG CAC-3′(SEQ ID No 11) targets the site 233 of the human Smad7 m-RNA. This is amixed-backbone oligonucleotide wherein the cytosine in the position 1and 14 of SEQ ID No 10 were replaced by 5-methylcytosine.

The Smad7 antisense ODN sequence 5′-GTMe-dCGCCCCTTCTCCCMe-dCGCAG-3′ (SEQID No 12) targets the site 403 of the human Smad7 m-RNA. This is amixed-backbone oligonucleotide wherein the cytosine belonging to CpGpairs in the position 3 and 16 of SEQ ID No 1 were replaced by5-methylcytosine (indicated as Me-dC).

The Smad7 antisense ODN sequence 5′-GATCGTTTGGTCCTGAA-3′ (SEQ ID No 13)targets the site 299 of the human Smad7 m-RNA.

The Smad7 antisense ODN sequence 5′-ATCGTTTGGTCCTGAAC-3′ (SEQ ID No 14)targets the site 298 of the human Smad7 m-RNA.

The Smad7 antisense ODN sequence MePGTMe-dCGCCCCTTCTCCCMe-dCGCAMePGC(SEQ ID No 15) targets the site 403 of the human Smad7 m-RNA. This is amixed-backbone oligonucleotide wherein the cytosine belonging to CpGpairs in the position 3 and 16 of SEQ ID No 1 were replaced by5-methylcytosine (indicated as Me-dC). In addition, methylphosphonatelinkages were placed at one of the ends of the oligonucleotides and atthe guanine residue in position 20.

Induction of Colitis

Five to six-week old male SJL/J mice were maintained in a specificpathogen-free animal facility. For induction of colitis, 2.5 mg TNBS (pH1.5-2.0; Sigma Aldrich) in 50% ethanol was administered per rectum tolightly anesthetized mice through a 3.5 F catheter. The catheter tip wasinserted 4 cm proximal to the anal verge, then 100 ml of fluid(TNBS/ethanol) was slowly instilled into the colon.

To ensure distribution of the TNBS within the entire colon and cecum,mice were held in a vertical position for 30 seconds after theinjection. Some of the mice were administered with 50% ethanol aloneusing the same technique and were used as controls.

Histologic Assessment of Colitis

Tissues removed from mice at indicated times of death were fixed in 10%formalin solution (Sigma Aldrich), embedded in paraffin, cut into tissuesections and stained with hematossiline and eosine. Stained sectionswere examined for evidence of colitis using different criteria such asthe presence of lymphocyte infiltration, elongation and/or distortion ofcrypts, frank ulceration and thickening of the bowel wall.

The degree of inflammation on microscopic cross-sections of the colonwas graded from 0 to 4 as follows:

0: no evidence of inflammation;

1: low level of lymphocyte infiltration with infiltration seen in a <10%high-power field (hpf=high power field), no structural changes observed;

2: moderate lymphocyte infiltration with infiltration seen in <10-25%hpf, crypt elongation, bowel wall thickening which does not extendbeyond mucosal layer;

3: high level of lymphocyte infiltration with infiltration seen in<25-50% hpf, thickening of bowel wall which extends beyond mucosallayer;

4: marked degree of lymphocyte infiltration with infiltration seenin >50% hpf, high vascular density, crypt elongation with distortion,transmural bowel wall-thickening with ulceration.

Isolation of Lamina Propria Mononuclear Cells (LPMC) and Treatment ofCells with Smad7 Antisense ODN

The lamina propria mononuclear cells (LPMC) were isolated from colonicspecimens. The specimens were first washed in HBSS-calcium magnesiumfree (Hanks' balanced salt solution, Sigma-Aldrich) and cut into 0.5-cmpieces. They were then incubated twice, each time for 15 minutes in HBSScontaining EDTA (0.37 mg/ml) and dithiothreitol (0.145 mg/ml) at 37° C.The tissues were then digested in RPMI containing collagenase D (400U/ml, Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and DNase I(0.01 mg/ml, Boehringer Mannheim Biochemicals, Indianapolis, Ind.) in ashaking incubator at 37° C.

The LPMC released from the tissue were resuspended in 100% Percoll,layered under a 40% Percoll gradient (Pharmacia Biotech AB, Uppsala,Sweden), and spun at 1,800 rpm for 30 minutes to obtain thelymphocyte-enriched population.

To assess the in vitro efficacy of Smad7 antisense ODN, the LPMCisolated from TNBS-treated mice, were resuspended in RPMI 1640(Sigma-Aldrich) supplemented with a serum replacement reagent HL-1(Biowhittaker) at a final concentration of 1×10⁶/ml in 24 well plates.For transfection of antisense ODN, 2 μl of lipofectamine 2000 reagent(LF, Invitrogen Italia SRL, San Giuliano Milanese) was used for each mlof cell medium following the protocol. Then, 2 μg/ml of antisense ODNand LF were combined and allowed to incubate for 20 minutes at roomtemperature. The obtained mixture was then added directly to the cells.After overnight culture, the cells were removed from the plate and usedfor analysis of Smad7 by Western blotting.

Treatment of Mice with Smad7 Antisense ODN

Two days after treatment with TNBS, mice were administered per rectum150 μg of each Smad7 antisense or sense oligonucleotide. At least 5 micefor group were examined. At fifth day mice were sacrificed and wholeintestinal mucosal samples were taken and analysed for Smad7 and Smad3content by Western blotting. In addition intestinal mucosal inflammationdegree entity was evaluated.

Protein Extraction and Western Blot Analysis

Both lamina propria mononuclear cells and whole colonic specimens werehomogenized using the above procedure. Then Smad7 expression wasrevealed by Western blotting.

At the end, the blots were stripped using a commercially availablesolution (Pierce) and probed with anti-actin antibodies (Sigma-Aldrich)to verify the same amount of protein were filled in each well. Detectionwas accomplished using a chemiluminescence kit (Pierce). The intensityof bands was analysed by a densitometer.

Both LPMC and whole colonic specimen samples proteins were also analyzedfor the content of phosphorylated and total Smad3 protein by Westernblotting using specific commercially available antibodies (Santa Cruz).

For the analysis of phosphorylated Smad3 a specific rabbit anti-humanantibody capable to recognize phosphorylated Smad2/3 proteins as antigen(1:500 final dilution), and a goat anti-rabbit antibody conjugated tohorseradish peroxidase (1:20.000 dilution) were used. Immunoreactivitywas visualized with a chemiluminescence kit (Pierce).

After detection, blots were stripped using a commercially availablesolution (Pierce) and incubated with a specific goat anti-human Smad3antibody (1:500 final dilution) followed by a rabbit anti-goat antibodyconjugated to horseradish peroxidase (1:20.000 dilution); thenimmunoreactivity was visualized with the above mentionedchemiluminescence kit (Pierce).

Test ELISA

The amount of active TGF-β1 was determined in the intestinal mucosalsamples. To this aim total proteins were extracted from mucosal samplesfrom mice with or without TNBS-induced colitis as above indicated. Thelevels of active TGF-β1 were analyzed using a commercially availableELISA kit (R&D Systems, Space Import-Export Srl, Milano). Opticaldensity was measured on a Dynatech MR 5000 ELISA reader at a wavelengthof 490 nm. Data were expressed as pg/100 μg of total proteins.

Results

After receiving TNBS mice developed diarrhoea and weight loss byevidence of the induction of colitis. The colon was macroscopicallyenlarged and histological analysis of its mucosa showed moderate tosevere inflammatory lesions.

To examine if induction of TNBS-colitis was associated with changes inthe production of TGF-β1, colonic specimens were taken from mice with orwithout colitis and analyzed for the content of active TGF-β1 by ELISA.

As several cell types which have the potential to synthesize TGF-β1 arepresent at intestinal level, it was evaluated in whole intestinalmucosal rather than LPMC samples.

In the absence of colitis low levels of active TGF-β1 were detected(85±12 and 94±26 pg/μg of total protein in unstimulated and controlsmice respectively). Significantly enhanced TGF-β1 levels were measuredin mucosal samples from mice with TNBS-induced colitis (985±120 pg/μg oftotal protein) (p<0.01). Even though this result seems to suggest thatduring TNBS-induced colitis there could be an increasing TGF-β1activity, the analysis of intracellular levels of active Smad3 inintestinal LPMC isolated from mice with colitis surprisingly revealed areduced Smad3 phosphorylation that was associated with an enhancedexpression of Smad7 (FIGS. 2 and 3). In particular, FIG. 2 illustratesthe presence of a band corresponding to the active (phosphorilated)Smad2/3 in LPMC isolated from the unaffected intestine but not from micewith TNBS-induced colitis. In the FIG. 3 it has been showed that the twobands, the lower 47 Kda band corresponding to the free Smad7 and theupper 102 Kda band corresponding to the TGF-β1 R1-Smad7 complex, arepresent only in LPMC specimens isolated from the intestine of mice withTNBS-induced colitis. These data indicate that local inflammationstimulates the synthesis of TGF-β1 which is not able to activate Smadsignalling and dampen the mucosal inflammation.

According to the present invention it has been evaluated if treatingTNBS mice with Smad7 antisense ODN could restore the endogenous TGF-β1function and limit the ongoing inflammation.

First, it has been tested the efficacy of the above mentioned Smad7antisense ODN (SEQ ID No 1 and SEQ ID No 4-15) to decrease Smad7expression both in vitro and in vivo experiments.

As regards in vitro experiments, the LPMC isolated from the intestine ofmice with TNBS-induced colitis were transfected with each of the Smad7antisense ODN and incubated overnight. Smad7 analysis was carried out byWestern blotting.

As regards in vivo experiments TNBS-treated mice were administered withSmad7 antisense ODN and after 3 days animals were sacrificed, tissuespecimens were taken and Smad7 analysis was carried out by Westernblotting.

Table 4 summarizes the results of these experiments and shows thepercentage inhibition obtained by each Smad7 antisense oligonucleotideboth in vitro and in vivo experiments. Data indicate mean±standarddeviation (SEM) of four separate in vitro experiments and mean±SEM offive separate in vivo experiments.

TABLE 4 Sequence % inhibition % inhibition SEQ ID (5′ → 3′) Site in LPMCin vivo No gtcgccccttctccccgcagc 403 29 ± 3 33 ± 0.5 1MePgtMedcgccccttctcc 403 34 ± 1.5 55 ± 3 4 cMe-dcgcaMePggtt tgg tcc tga aca tgc 294 26 ± 2.6 25 ± 3.4 5 gtt tgg tcc tga acat 29616 ± 2 15 ± 3.2 6 gtt tgg tcc tga acatg 295 17 ± 3.1 10 ± 1.12 7agc acc gag tgc gtg 577 (*) 27 ± 0.88 25 ± 2.7 8 agc MePagcacMedc gag577 (*) 29 ± 1.65 30 ± 1.3 9 tg Medc gtg agcMeP cga aca tga cct ccg233 (**) 33 ± 2.3 32 ± 1.89 10 cac Me-d cga aca tga cct 233 (**) 36 ±1.5 34 ± 2.2 11 cMe-d cg cac gtMedcgccccttctcccMe 403 32 ± 4.1 42 ± 1.812 dcgcag gatcgtttggtcctgaa 299 13 atcgtttggtcctgaac 298 14MePgtMedcgcccottctcc 403 34 ± 1.6 56 ± 3 15 cMedcgcaMePgc (*) SequencesNo 16 and (**) No 12 of the Patent U.S. Pat. No. 6,159,697 by ISIS.

All the antisense ODN were effective in reducing Smad7 expression whentransfected in vitro in LPMC isolated from TNBS-treated murine models.From the analysis of the value of percentage inhibition shown in Table 4it is remarkable that antisense oligonucleotidic sequences SEQ ID No 4,10, 11, 12 and 15 showed the major efficacy.

Nevertheless, the percentage of Smad7 expression inhibition obtained byin vivo treatment with oligonucleotidic sequences SEQ ID No 10 and 11did not significantly differ from that documented in vitro experiments.

Instead, treatment of mice with antisense ODN SEQ ID No 4 and 12 and 15resulted clearly in a greater percentage of Smad7 inhibition than thatobtained in vitro experiments, that is 55% vs 34%, 42% vs 32% e 56% vs34% respectively (P<0.01).

In contrast, treatment of mice with antisense oligonucleotide SEQ ID No7 caused a reduction in Smad7 expression in vivo which was of lowerentity than that resulting when the antisense oligonucleotide wastransfected in LPMC in vitro, that is 10% vs 17%, P<0.01.

Overall, these results suggest that only specific modifications into aSmad7 antisense ODN sequence are able to improve its pharmacokinetic,biochemical and biophysical profile.

No sign of acute toxicity was documented in mice receiving antisenseoligonucleotides (SEQ ID No 1 and SEQ ID No 4-15). One out of 5, treatedwith TNBS, died after 3 days (20%). Similarly, ⅕ of mice receiving theSmad7 sense oligonucleotide died after 4 days.

No mortality was documented in mice group treated with Smad7 antisenseODN SEQ ID No 1 and SEQ ID No 4-15.

The use of antisense ODN sequences SEQ ID No 13 and SEQ ID No 14 isassociated with a reasonable in vitro inhibition activity (11% and 9.5%,respectively). Nevertheless, the in vivo administration of suchsequences was unexpectedly joined with a marked deterioration of thecolitis, up to cause the death of all the mice after 72 hours oftreatment.

Macroscopic analysis of the intestinal samples taken from these mice hasrevealed the presence of a severe colitis and this was associated to asubstantial increase in the intestinal Smad7 expression.

As above said, it was tested the efficacy of Smad7 antisense ODN tolimit the ongoing inflammation. For this purpose, mice after inductionof colitis were administered with antisense oligonucleotides SEQ ID No1, 4, 5 and 15 considering 5 animals for each group.

Following the treatment with Smad7 antisense ODN it has been revealed areduction of the mucosal inflammation. This result was particularlyevident in mice treated with antisense oligonucleotides 4 and 15.Indeed, the colitis severity of grade 3-4 in mice with colitis notreceiving antisense reached grade 2 or 3 after administration ofantisense oligonucleotide sequences 1 or 5 respectively, while in micetreated with oligonucleotidic sequences 4 or 15, inflammation has notexceeded grade 1.

To examine if Smad7 antisense oligonucleotides were effective also whenadministered orally, mice with TNBS-induced colitis were treated the dayafter the induction of colitis with Smad7 antisense oligonucleotide 4 or15 or control (sense).

For this purpose oligonucleotides were risuspended in a bicarbonatesolution. The final volume of the solution administered to each mousewas of 350 μl and contains doses of oligonucleotide equivalent to 250,500 or 1000 μg. Such solution was administered per os through acatheter.

At fifth day mice were sacrificed and analysis of Smad7 expression andof inflammation degree were evaluated as indicated in the previousparagraphs. All the mice treated with antisense oligonucleotide, and notwith the control sense oligonucleotide, showed a meaningful reduction ofSmad7 expression ad an increased Smad3 phosphorylation, independentlyfrom the dose of the oligonucleotide being used.

Substantially, Smad7 inhibition was associated with a weight recovery asshown in FIG. 4. The FIG. 4 exhibits a graph which shows the percentagechange in weight of the mice with TNBS-induced colitis treated or nottreated with Smad7 antisense oligonucleotide (SEQ. N. 15) or control(sense). Both oligonucleotides were administered per os at the dose of250 μg through catheter two days after the induction of colitis. Theweight loss documentable at the second day in each of the three groupsindicates that the treatment with TNBS induced colitis. Further it wasproved that starting from the fourth day mice treated with Smad7antisense oligonucleotide, but not with the control, showed a bodyweight recovery. The apparent and slight recovery seen at the fifth dayin mice with TNBS-induced colitis is due to the fact that the 21.4% ofmice with colitis died at the fourth day and therefore they were notconsidered in the evaluation of the body weight at the fifth day.

Smad7 inhibition was correlated to a marked suppression of tissutalinflammation as shown in FIGS. 5 and 6. FIG. 5 exhibits the images ofthe colon extracted from a mouse with TNBS colitis and from a mouse withTNBS colitis treated with Smad7 antisense oligonucleotides (SEQ. ID No15). The oligonucleotide was administered per os at the dose of 250 μgthrough a catheter, at the second day after the induction of colitis. Ithas been showed that the colon from the mouse with TNBS-colitis ishighly inflammed, shortened and thickening. On the contrary, the mousereceiving Smad7 antisense shows a colon of normal length and thicknessand no macroscopic signs of phlogosis. FIG. 6 exhibits histologicalaspect of a colon section from a mouse without colitis or from mice withTNBS-colitis treated or not treated with Smad7 antisense oligonucleotide(SEQ ID No 15) or the control (sense). Both oligonucleotides wereadministered per os at the dose of 250 μg through catheter the secondday after the induction of colitis. It has been shown that in the mousewithout colitis, glands appear rectilinear and uniform with a normalcontent of muciparous cells and inflammatory elements of lamina propria.On the contrary, in the colon of TNBS treated mice receiving or not thecontrol oligonucleotide, there was a total destruction of the glandularstructure, with a muciparous and a massive inflammatory cellsinfiltration in the lamina propria. In the colic section of the mousetreated with TNBS and receiving Smad7 antisense oligonucleotide thepresence of a normal glandular structure and the absence of phlogosiswere demonstrated.

Together these observations suggest that the use of antisense ODN, whichshow the higher efficacy of Smad7 inhibition accompanied by the absenceof side effects, following the in vivo administration, can represent apromising therapeutic strategy in the control of mucosa inflammationduring IBD, especially if such characteristics of efficacy and toxicitywere compared with the results achieved with other antisense ODNsequences with the same efficacy in the Smad7 in vitro inhibition.

EXAMPLE 3: IN VIVO AND IN VITRO STUDIES ON THE EFFECTS OF THEADMINISTRATION OF SMAD7 ANTISENSE OLIGONUCLEOTIDE (SEQ ID NO:16) IN THECONTEXT OF ACUTE (TNBS- AND OXAZOLONE-COLITIS), RELAPSING (TNBS) ANDLONG TERM (SCID TRANSFER COLITIS) INFLAMMATION IN MICE

In the present experiment the inventor examined TGF-β1 signaling in thecontext of acute (TNBS- and oxazolone-colitis), relapsing (TNBS) andlong term (SCID transfer colitis) inflammation in mice. In theseconditions it was found that induction of colitis is accompanied byabundant local TGF-β1 production associated with high Smad7 that, as inhumans, blocks TGF-β1 signaling. However, administration of Smad7anti-sense oligonucleotides, such as GTXGCCCCTTCTCCCXGCAGC, wherein X is5-Me-dC (5-methyl 2′-deoxycitidine 5′-monophosphate) (SEQ ID NO:16)relieves the Smad7 block and enables TGF-β1 signaling and ameliorationof disease. In fact, with respect to TGF-β1 signaling and Smad7expression, acute and relapsing models of colitis reproduce the findingsobserved in humans IBD and that Smad7 anti-sense oligonucleotide maytherefore have an important place in IBD therapy.

Materials and Methods

Induction of TNBS- and Oxazolone-Colitis

Studies of acute hapten-induced colitis were performed in 5-6 week oldmale SJL mice (Harlan Laboratories, S. Pietro al Natisone, UD, Italy),and maintained in the animal facility at the Istituto Superiore diSanità, Rome, Italy. All studies were approved by Animal Care and UseCommittee of Istituto Superiore di Sanità and Italian Ministry ofHealth. For induction of colitis, 2.5 mg of TNBS or 6 mg of oxazolone(Sigma-Aldrich, Milan, Italy) in 50% ethanol was administered to lightlyanesthetized mice through a 3.5 F catheter inserted into the rectum. Thecatheter tip was inserted 4 cm proximal to the anal verge, and 150 μl offluid was slowly instilled into the colon, after which the mouse washeld in a vertical position for 30 seconds. Controls consisted of micetreated with 150 □l of 50% ethanol and untreated naive mice. Weightchanges were recorded daily to assess the induction of colitis andtissues were collected for histologic study and protein analysis. Forhistological analysis tissues were fixed in 10% neutral bufferedformalin solution, embedded in paraffin, cut into tissue sections andstained with hematoxylin and eosin (H&E). For TNBS-colitis stainedsections were examined for evidence of colitis and assigned a colitisscore by considering the presence of acute and chronic inflammatoryinfiltrates, elongation and/or distortion of crypts, frank ulceration,and thickening of the bowel wall, as described elsewhere (Goreli L, etal., 2002). For oxazolone-induced colitis, stained sections wereexamined and assigned a colitis score by examining the slide for thepresence of hypervascularization, mononuclear cells, epithelialhyperplasia, epithelial injury, and granulocytes (Boirivant M, et al.,1998; Boirivant M, et al., 2001; Lawrance I C, et al., 2003).

Colitis was assessed as described elsewhere (Lawrance I C, et al.,2003). Briefly, serial paraffin sections of the colon were stained withH&E, and the degree of inflammation was scored as absent, mild,moderate, or severe based on the density and extent of both the acuteand chronic inflammatory infiltrate, loss of goblet cells, and bowelwall thickening. An inflammatory infiltrate of low cellularity confinedto the mucosa was scored as mild inflammation, and transmuralinflammation with extension into the pericolonic adipose tissue withhigh cellularity was scored as severe. Intermediate changes were scoredas moderate inflammation. SCID model of colitis: colitis was induced inSCID mice according to the protocol described by Read and Powrie (ReadS, et al., 1999). Development of colitis was monitored by weekly recordof the body weight and presence of loose stools. Diagnosis of colitiswas made by histological analysis of the colon.

Inhibition of Murine Smad7 Production by Smad7 Antisense Oligonucleotide

To evaluate the ability of Smad7 antisense to inhibit Smad7, murinemacrophage cell lines (RAW cells) were transfected with Smad7 antisenseor sense oligonucleotides (2 μg/ml; GTXGCCCCTTCTCCCXGCAGC, wherein X is5-Me-dC (SEQ ID NO:16)) by lipofectamine for 48 hours. The cells werethen harvested and subjected to Western blot analysis of Smad7. Theefficiency of the transfection was determined with afluorescein-labelled Smad7 antisense oligonucleotide and transfectionrate was evaluated by performing flow cytometry and fluorescentmicroscopy 24 hours after transfection. The same fluorescein-labelledSmad7 antisense oligonucleotide was used to assess the in vivo uptake ofthe orally administered oligonucleotide. To this end, mice withTNBS-colitis were given fluorescein-labelled antisense DNA (125μg/mouse) by oral administration 1 day after TNBS injection and thensacrificed 0, 4, 8, 16, 24 and 48 hours later. At each time point,stomach, small intestine, colon, liver, spleen, and kidney werecollected, cut in sections and analyzed by fluorescent microscopy.

To examine the therapeutic effect of Smad7 antisense oligonucleotide(GTXGCCCCTTCTCCCXGCAGC, wherein X is 5-Me-dC (SEQ ID NO:16)) on thecourse of ongoing intestinal inflammation, mice were treated with asingle dose of Smad7 antisense or sense oligonucleotide (from 50, 125 or250 μg/mouse) in 500 μl of bicarbonate solution (pH 9.5) by oraladministration on the day after the induction of colitis. The mice werethen monitored daily for weight changes and then sacrificed 3(TNBS-colitis) and 2 (oxazolone colitis) days after oligonucleotideadministration. To evaluate the effect of Smad7 antisenseoligonucleotide administration on relapsing TNBS-colitis in BALB/c mice,mice were divided into two groups the day after the final TNBSadministration: one group was treated with Smad7 antisenseoligonucleotide and the other with sense oligonucleotide (125 μg/mouse,on days 1 and 3 after the last TNBS dose). Weight changes were recordeddaily and mice were sacrificed at day 6.

To evaluate the effect of Smad7 antisense oligonucleotide (SEQ ID No:16)on SCID model of colitis, 6 weeks after cell transfer the mice weretreated with oral Smad7 anti-sense or sense oligonucleotide (125μg/mouse) and weight changes were recorded on day 1, 3, 7 and 10 aftertreatment.

Isolation of Lamina Propria Mononuclear Cells (LPMC)

LPMC were isolated as described in the previous example or by cellsorting after staining cells with anti-mouse CD3-FITC (BD Pharmingen)using a FACS-ARIA. Purity of CD3+ cells was confirmed by flow cytometryand was consistently higher than 95%.

Determination of Cytokine Secretion by ELISA

Total protein extracts were prepared as previously described in theabove examples. Briefly, snap frozen mucosal samples or cells werehomogenized in buffer containing 10 mM Hepes (pH 7.9), 10 mM KCl, 0.1 mMEDTA, and 0.2 mM EGTA, supplemented with 1 mM dithiothreitol (DTT), 10μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM phenylmethanesulphonylfluoride (Sigma).

Western Blotting

Western blotting for the detection of p-Smad3, Smad7 were performed asabove described.

Statistical Analysis

The significance of differences between groups was determined usingeither the Mann-Whitney U test or Student t test.

Results

It has been shown that TNBS-colitis is characterized by decreasedp-Smad3 and increased Smad7 expression. Particularly, levels ofactivated TGF-β1 were higher in extracts from mice with TNBS-colitis(median: 56.5; range: 44-83 μg/100 μg total proteins) than those fromuntreated (median: 8.2; range: <10-19 μg/100 μg total proteins, p=0.03)or ethanol-treated mice (median: 19; range: <10-28 μg/100 μg totalproteins, p=0.04). Since activated TGF-β1 is generated in theextracellular space, this indicates that increased amounts of TGF-β1 issecreted as well as synthesized in TNBS-colitis.

In a complementary analysis, TGF-β1 was analyzed in extracts ofepithelial cells. Colonic epithelial cells from mice treated with TNBScontained higher levels of TGF-β1 (44±7 pg/100 μg total proteins) thanthose measured in extracts of ethanol-treated mice (35±2.5 pg/100 μgtotal proteins), but the difference was not statistically significant.The lack of statistical significance could rely on the fact that theisolated colonic cells undergo rapid apoptosis. Taken together, thesedata show that TNBS-colitis is associated with a significant increase inTGF-β1 production that is, at least in part, derived from epithelialcells.

Administration of Smad7 Antisense Oligonucleotide Inhibits Smad7 ProteinExpression and Restores TGF-β1 Signaling in TNBS-Colitis

The above data prompted us to explore the possibility that inTNBS-colitis, high local Smad7 blocks the immunosuppressive activity ofthe endogenous TGF-β1 and thus contributes to ongoing intestinalinflammation. Thus, it was determined whether the administration ofSmad7-specific antisense oligonucleotide (GTXGCCCCTTCTCCCXGCAGC, whereinX is 5-Me-dC (SEQ ID NO:16)) could affect the course of experimentalcolitis. In these studies, RAW cells constitutively expressing Smad7were transfected with Smad7 antisense or sense oligonucleotides and thenassessed for Smad7 protein expression by Western blotting. Using afluorescein-labeled Smad7 antisense DNA to identify transfected cells byflow cytometry, almost ⅔ of the RAW cells were efficiently transfected(FIG. 7A). In addition, the transfected cells exhibited a markeddecrease in Smad7 expression (FIG. 7A, right inset). These results ledus to determine the ability of this oligonucleotide to inhibit Smad7 invivo. Initially, we administered fluorescein-labelled Smad7 antisenseoligonucleotide to mice with TNBS-colitis at day 1 after colitisinduction by oral gavage in order to determine the tissue distributionof oligonucleotide administered by this route. Administration by oralgavage was selected so as to target the mucosal tissues rather thansystemic tissues and thus to minimize potential adverse effects.Fluorescein-label oligonucleotide was seen in both the small intestinaland colonic lamina propria and epithelial layer at 4, 8 (FIG. 7B) and 16hours after oligonucleotide administration and then disappeared at 24and 48 hours after administration (not shown). Finally, it wasdetermined whether administration of Smad7 antisense oligonucleotideadministered by oral gavage affected Smad7 and p-Smad3 expression in thecolon of mice with TNBS-colitis. Administration of Smad7 antisense butnot sense oligonucleotide significantly reduced Smad7 (p=0.02) andenhanced p-Smad3 (p=0.03) (FIG. 7C).

Orally Administered Smad7 Antisense Oligonucleotide Ameliorates AcuteTNBS-Colitis

Therapeutic effectiveness of Smad7 antisense oligonucleotide(GTXGCCCCTTCTCCCXGCAGC, wherein X is 5-Me-dC (SEQ ID NO:16)) in micewith acute TNBS-colitis was assayed. Mice administered Smad7 antisenseoligonucleotide as a single oral dose of 125 or 250 μg/mouse (but not 50μg/mouse) on day 1 after induction of TNBS-colitis exhibited an initialweight loss which rapidly stabilized so that by day 4 the weight loss inthe treated group was clearly lower than in the untreated group (p<0.01)(FIGS. 8A and B), and by day 7 it was not different from the initialweight (not shown). In addition, as shown in FIG. 8C, histologicexamination of colon tissue as well as blinded histologic scoring ofcolitis in the different groups were significantly reduced inantisense-treated mice as compared to untreated or sense-treated mice(p<0.01). Then, it was determined the effect of orally administeredSmad7 antisense oligonucleotide (again given as a single 125 μg dose onday 1 after TNBS-colitis induction) on cytokine production in mice withTNBS-colitis. Administration of Smad7 antisense but not senseoligonucleotide to mice with TNBS-colitis significantly reduced thecolonic production of both IL-12 and IFN-γ (p<0.03) (FIG. 9 A, B, C).

Administration of Smad7 Antisense Oligonucleotide Ameliorates OxazoloneColitis

Next the effect of Smad7 inhibition on oxazolone colitis byadministration of Smad7 antisense oligonucleotide(GTXGCCCCTTCTCCCXGCAGC, wherein X is 5-Me-dC (SEQ ID NO:16)) wasdetermined. The oligonucleotides were administered by oral gavage 1 dayafter the induction of colitis, and mice were sacrificed at day 3.Analysis of Smad7 expression showed a marked reduction in the colon ofmice treated with the antisense but not sense oligonucleotide, and thiswas associated with increased p-Smad3 (FIGS. 10A and B, p=0.03).Moreover, mice treated with the Smad7 antisense oligonucleotide exhibitless weight loss and less mortality than those treated with senseoligonucleotide: the mean weight observed at day 3 was 90.3%±2.8 and84.7%±1.7 of baseline in mice treated with the antisense or senseoligonucleotide respectively and corresponding mortality figures were 0%and 21.4% of mice. These data were consistent with histologic study oftissues in that Smad7 antisense but not sense oligonucleotide led tomarkedly decreased inflammation (FIG. 10C).

Smad7 Antisense Oligonucleotide Reverses Relapsing TNBS-Colitis

In further studies, we examined whether the Smad7 antisenseoligonucleotide (GTXGCCCCTTCTCCCXGCAGC, wherein X is 5-Me-dC (SEQ IDNO:16)) could reverse relapsing colitis by the use of a TNBS-colitismodel. In this model the mice lost weight during the first 1-2 daysfollowing each TNBS instillation but then regained the same or moreweight before the next instillation a week later; thus, the averageweight of mice in the group administered TNBS was similar to those inthe group not administered TNBS at the time of fourth and finaltreatment. It should be noted that although the mice in this model hadpersistent inflammation, the latter was being maintained by weekly TNBSadministration and thus could be considered as a colitis characterizedby persistent inflammation with recurrent relapses of acuteinflammation. Histological examination of the colons obtained from miceone day after the fourth weekly instillation of TNBS revealed that 15%of mice developed a severe inflammation, while 70% and 15% of mice had amoderate or mild colitis respectively (FIG. 11A). In addition, Westernblot analyses of extracts from the inflamed colons of TNBS-treated miceexhibited reduced p-Smad3 and increased Smad7 as compared with extractsof control mice (FIG. 11B). Up-regulation of Smad7 was seen both in CD3+and CD3-negative LPMC isolated from mice with established TNBS-inducedcolitis (FIG. 11B, right inset). Finally, to examine if treatment ofmice with Smad7 oligonucleotide could reverse the mucosal inflammationpresent after the fourth instillation of TNBS, Smad7 antisenseoligonucleotide was administered to mice by oral gavage on days 1 and 3after the last (4^(th)) TNBS instillation. Mice treated with Smad7antisense oligonucleotide lost only 2% of body weight by day 2 after thefirst Smad7 antisense oligonucleotide administration, regained all ofthe weight lost by day 3, and were above baseline weight by day 6. (FIG.11C). In contrast, mice treated with sense oligonucleotide exhibited asignificantly greater loss of weight and did not fully reach baselineweight by day 6 (FIG. 11C). In mice treated with the above antisenseoligonucleotide, Smad7 was reduced both in CD3+ and CD3− negative LPMC(FIG. 7C, right inset). Histological examination of colons of micetreated with anti-sense oligonucleotide showed that 45% of miceexhibited mild colitis while 55% of them had no evidence ofinflammation. In contrast, all mice treated with sense oligonucleotidesexhibited moderate (80%) or mild (20%) colitis (FIG. 11D). These datasuggest that Smad7 anti-sense oligonucleotide can interrupt and reversethe course of a relapsing colitis.

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The invention claimed is:
 1. An antisense oligonucleotide against SMAD7comprising SEQ ID NO: 11 (5′-XGAACATGACCTCXGCAC-3′), wherein X is5-methyl-2′-deoxycytidine 5′-monophosphate.
 2. The antisenseoligonucleotide of claim 1, wherein at least one internucleoside linkageis a phosphorothioate linkage.
 3. A method of treating ulcerativecolitis, comprising administering to a patient in need thereof aneffective amount of the antisense oligonucleotide of claim
 1. 4. Amethod of treating inflammatory bowel disease, comprising administeringto a patient in need thereof an effective amount of the antisenseoligonucleotide of claim
 1. 5. A pharmaceutical composition comprisingthe antisense oligonucleotide of claim 1 and a pharmaceuticallyacceptable adjuvant and/or excipient.
 6. The pharmaceutical compositionof claim 5, wherein the pharmaceutical composition is an oralpharmaceutical composition.